Method and device for studying transport of an agent across a bilayer membrane in bioanalytical sensor applications

ABSTRACT

The present invention provides a method for studying transport of an agent across a membrane comprising the steps a) providing at least one surface with a bilayer structure tethered to the surface, said bilayer structure comprising a detection volume, b) contacting the bilayer with at least one agent to be analysed, and c) detecting a change in refractive index in the detection volume resulting from transportation of the agent across the membrane. Further there is provided a device comprising a) at least one surface, b) at least one bilayer structure tethered to the surface, and c) at least one sensor capable of detecting a change in refractive index in a detection volume, wherein the bilayer structure encloses a first volume of the detection volume and wherein the volume not enclosed by the bilayer structure but within the detection volume is a second volume and wherein the ratio between the first volume and second volume is above about 0.001.

The present invention relates to a method and a device for themeasurement of transport of an agent across a bilayer membrane.

BACKGROUND

Transport across membranes and the influence of molecules associatedwith a membrane such as membrane proteins are important to study forinstance when developing new drugs and also in many other contexts.

In 1895 Overton suggested that molecules permeate the cells in the samerelative order as their oil-water partition coefficient [1] and in 1943Danielli proposed that a continuous lipid bilayer acts as a diffusionbarrier determining the rate of passive diffusion across cellularmembranes [2]. However, the first means to directly study permeationacross an isolated lipid bilayer became available in the early 60-iesthrough the development of means to prepare single lipid bilayersseparating two aqueous compartments [3]. While this technique is wellsuited for permeability studies of charged solutes, the standardapproach of today for studies of passive and active transport ofnon-electrolytes, including water transport and drug uptake, relies onmeasurements of osmotic-induced size changes of suspended lipid vesicles(so called liposomes) [4]. This method is based on dynamic lightscattering (DLS) measurements of variations in scattered light intensityas the liposome dimension changes in response to osmotically inducedwater transfer across the lipid membrane, upon which the liposomes first(<1 ms) shrink, as water diffuse out of the liposomes, and subsequentlyswell as water reenters the liposomes driven by the inward permeation ofsolute molecules [5]. Although successfully applied in numerous studieson the nature of passive and active solute permeation [6], the method isrestricted by the fact that a change in liposome size is an indirecteffect, which does not necessarily correlate with the actual solutetransport. In addition, since not only the liposome size, but alsoliposome motion, solute refractive index, and membrane aggregationcontribute to the intensity of the scattered light, the quantificationof solute transfer is not always straightforward [7,8]. From a practicalperspective, this methods also suffers from low signal-to-noise ratios,which means that averaging from multiple data series is generallyrequired to resolve kinetic traces. Furthermore, since measurements areperformed on suspended liposomes, the method is not compatible withparallel or sequential screening of the very same liposome sample. This,in turn, means that substantial amounts of material are generallyneeded. Besides somewhat improved sensitivities, these limitations holdsalso for a less wide-spread method, in which osmotically inducedliposome size fluctuations are recorded by monitoring changes inself-quenching of liposome-entrapped fluorophores upon liposomeshrinkage and concomitant increase in fluorophore concentration [9]. Thepossibility to screen multiple recognition events either sequentially orsimultaneously is one of the main advantages of surface-basedbioanalytical sensor technologies, where the very same set ofsurface-immobilized probe molecules is exposed to a series of differentcompounds in an automated fashion. An additional reason for the emergingimportance of these sensors in life-science stems from the fact thatthey can be combined with micro-fluidic handling [10], which makes themcompatible with small sample volumes and thus well suited formeasurements on rare and non-abundant substances. Surface plasmonresonance (SPR) is today the dominating surface-based bioanalyticalsensor. It is based on excitation of laterally propagating surfaceplasmons at planar metal (usually gold) substrates, where the conditionfor SPR excitation is extremely sensitive to changes in interfacialrefractive index, Δn_(interface), induced by for example biomolecularbinding within a region in close proximity to the surface (typicallyhundreds of nanometers). Hence, by immobilizing probe molecules on thesurface, binding of targets to the immobilized probes can be monitoredin real time via a response which to a good approximation isproportional to Δn_(interface) [11, 12].

Other known devices for studying transport across membranes includeliposomes in a solution. To the liposome membrane there are associatedfor instance a membrane protein of interest and the membrane proteinmediated transport of an agent across the membrane is studied usingmethods involving for instance fluorescence and/or radioactivitymeasurements.

US 2004/0033624 disclose a membrane receptor reagent and assay device.Liposomes are tethered to a surface with anchor groups. The surfacecomprises reagent ligands that are tethered to the surface. The ligandsare able to bind reversibly to a receptor in the liposome membrane. Themembrane protein in the liposome is associated with a molecule with thecapability to be excited by emitted energy from the surface and therebyproduces a detectable signal. The binding of the membrane protein in theliposome membrane to the reagent ligands on the surface tend to pull theliposomes towards the surface. A test molecule with affinity to themembrane protein will bind competitively to the membrane protein and tosome extent replace the reagent ligand on the surface. This will resultin that membrane proteins come off the surface and are redistributed inthe liposome membrane, while the liposome still is anchored to thesurface. The membrane proteins will have a larger average distance fromthe surface and thereby they receive less energy from the surface, sincethe energy transfer from the surface is distance dependent. This can bedetected. The method in US 2004/0033624 requires some kind of label suchas a fluorophore in order to function.

In the assays for the measurement of membrane protein mediated transportacross a membrane according to the state of the art, there is room foran improvement regarding the amount of membrane protein that has to beused. Membrane proteins are often difficult and expensive to purify insubstantial quantities.

In the state of the art there is also room for improvement because alabel has to be used in many assays. Labelling is not always possiblesince it may alter the structure and function of the analyte/receptorand interfere with the molecular interaction that is to be investigated.In addition fluorescent markers are hydrophobic, which can causeunspecific background binding.

SUMMARY OF THE INVENTION

It is an object of the present invention to address at least some of thedisadvantages associated with known analysis methods and devices formeasuring transport across membranes, and to provide an improved methodand device, alleviating at least some of the problems in the prior art.Further disadvantages associated with known methods and devices and theadvantages associated with the embodiments of the present invention willbe apparent to a skilled person upon a closer study of the description,example and claims.

There is disclosed a method and device as defined in the claims,incorporated herein by reference.

Further aspects of the invention, as well as their advantages, willbecome evident to the skilled person upon closer study of thedescription, example, claims and drawings.

DEFINITIONS

Before the present device and method are described in detail, it is tobe understood that this invention is not limited to the particularconfigurations, method steps, detection methods, transducing methods,sensors and materials disclosed herein as such configurations, methodsteps, detection methods, transducing methods, sensors and materials mayvary somewhat. It is also to be understood that the terminology employedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims and equivalentsthereof.

It must also be noted that, as used in this specification and theappended claims, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, a reference to a reaction mixture containing “an analyte”includes a mixture of two or more analytes.

The term “about” when used in the context of numeric values denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. Said interval is preferably ±10%.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outherein.

The term “bilayer structure” as used throughout the description and theclaims denotes a double layer structure of atoms or molecules andespecially lipids. The term encompasses bilayers of all geometriesincluding but not limited to curved bilayers. Examples of bilayerstructures include but are not limited to liposomes.

The term “detection volume” as used throughout the description and theclaims denotes a volume in which the refractive index is measured.

The term “ellipsometry sensor” as used throughout the description andthe claims denotes a sensor comprising an ellipsometer.

The term “ionophore” as used throughout the description and the claimsdenotes a lipid-soluble molecule with the capability to transport ionsacross a lipid bilayer.

The term “lipid” as used throughout the description and the claimsdenotes any fat-soluble molecules. Examples of lipids include but arenot limited to; fats, oils, waxes, cholesterol, sterols, monoglycerides,diglycerides, and phospholipids.

The term “liposome” as used throughout the description and the claimsdenotes an essentially spherical vesicle comprising a lipid bilayermembrane. Liposomes may comprise a core of an aqueous solution. Thelipid membrane of the liposome may comprise components such as but notlimited to proteins, glycolipids, steroids and other membrane-associatedcomponents.

The term “membrane” as used throughout the description and the claimscomprises all types of membrane such as but not limited to a bilayermembrane. A membrane may comprise molecules such as but not limited toproteins and lipids.

The term “membrane protein” as used throughout the description and theclaims denotes a protein which is associated with a membrane.

The term “sensor” as used throughout the description and the claimsdenotes a transducer which uses a type of energy, a signal of some sort,and converts it into a reading for the purpose of information transfer.

The term “spacer” as used throughout the description and the claimsdenotes a molecule that is used to link together other molecules so thatthere is a space between the linked molecules.

The term “surface” as used throughout the description and the claimsshould be interpreted in a wide sense. A surface can in the presentinvention be used to support means on which structures can be tethered.

The term “surface plasmon resonance sensor” as used throughout thedescription and the claims denotes a sensor utilising the excitation ofsurface plasmons by light.

The term “tether” as used throughout the description and the claimsdenotes the attachment or entrapment of a material to a surface in amanner that confines, but not necessarily restricts the movement of thematerial.

DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a method for studyingtransport of an agent across a membrane comprising the steps

-   -   a) providing at least one surface with a bilayer structure        tethered to the surface, said bilayer structure comprising a        detection volume,    -   b) contacting the bilayer with at least one agent to be        analysed, and    -   c) detecting a change in refractive index in the detection        volume resulting from transportation of the agent across the        membrane.

The bilayer structure is in contact with a solvent. In one embodimentthe solvent is water. The bilayer structure comprises at least onemembrane.

In one embodiment at least one membrane protein is associated with thebilayer structure. If it is desired to study transport across a membranewhich is mediated by a membrane protein, the membrane protein is in oneembodiment inserted in the bilayer membrane. Also transport of othermolecules on the transport across a membrane can be studied. Examplesinclude but are not limited to passive diffusion of molecules acrossmembranes and transport which is facilitated by for example ionophoresand permeation enhancers. A permeation enhancer is a molecule which isadded to the solution comprising the analyte and which influences thetransport across the membrane.

In one embodiment at least one entity from the group consisting of amembrane protein, and an ionophore is associated with the bilayerstructure.

The surface comprises a detection volume in which the refractive indexis measured. The bilayer structure tethered to the surface is entirelyor partly within the detection volume. In one embodiment the detectionvolume is a volume limited by the surface and a plane 250 nm from thesurface and parallel to the surface.

In one embodiment the sensitivity of the detection volume decreasesexponentially with the distance from the surface. In one embodiment thedetection volume is limited to a volume where the detection sensitivityis more than 1% of the maximum sensitivity at the surface. Thus it ispossible to calculate a detection volume also for an embodiment wherethe sensitivity of the detection volume decreases exponentially with thedistance from the surface. An example of an embodiment where thesensitivity of the detection volume decreases exponentially with thedistance from the surface includes but is not limited to an embodimentcomprising a surface plasmon resonance sensor.

The bilayer structure is such that an analyte can not diffuse or movefreely across the bilayer structure because of the bilayer. However, theanalyte can still pass through the membrane.

In one embodiment the bilayer structure comprises a volume which is atleast partly enclosed by the bilayer.

The analyte to be investigated is in one embodiment added to the solventin contact with the bilayer structure. Depending on the properties ofthe analyte, it may be transferred very slowly or hardly at all acrossthe bilayer membrane. For some analytes, the analyte may be transferredacross the bilayer membrane at a higher rate. The transport of ananalyte across the membrane depends on the properties of the analyte andon the molecules in the bilayer.

To the bilayer structure there is in one embodiment associated at leastone molecule to be studied. Examples of such molecules include but arenot limited to; ionophores, integral membrane spanning proteins,proteins binding to one side of the bilayer through a hydrophobic patch,mainly hydrophilic proteins attaching hydrophobically through acovalently bound alkyl chain, mainly hydrophilic proteins with ahydrophobic pocket that can attract a lipid chain, and hydrophilicproteins binding electrostatically.

The present invention provides a possibility to study transport across abilayer membrane facilitated by a molecule. The transport of the analyteacross the bilayer membrane is measured by measuring the refractiveindex.

Examples of analytes include but are not limited to positive ions,negative ions, drug molecules, organic molecules, inorganic molecules,receptor ligands, sucrose, DNA, RNA, peptides, and proteins.

In one embodiment an analyte is added to the solvent in contact with thebilayer structure and is spread over a volume which is on one side ofthe bilayer in the bilayer structure. The analyte dissolved in thesolvent at a certain concentration has one refractive index. In thevolume on the other side of the bilayer in the bilayer structure theconcentration of the analyte may be different, which will give adifferent refractive index for that volume. By measuring the refractiveindex it is possible to monitor the transport across the membrane.

Advantages of surface-based techniques include that they better can becombined with micro-fluidic devices, they can be fully automated andmeasurements require smaller sample volumes

In one embodiment the sensor utilises a technique that measuresrefractive index changes in close proximity to the surface. In oneembodiment the sensor has the capability to measure changes inrefractive index in the interval 0-250 nm from the surface.

In one embodiment the method comprises detecting a change in refractiveindex with a sensor selected from the group consisting of a surfaceplasmon resonance sensor, an ellipsometry sensor, and an opticalwaveguide laser spectroscopy sensor.

In one embodiment the bilayer structure comprises at least one liposome.In a further embodiment the bilayer structure comprises at least onelayer of liposomes. In still a further embodiment the bilayer structureis a layer of tethered liposomes. In another embodiment the bilayerstructure comprises at least two layers of liposomes. In yet anotherembodiment the bilayer structure is a multitude of liposomes tethered tothe surface.

In one embodiment the liposomes are tethered to the surface with aspacer molecule. In one embodiment where there are several layers ofliposomes there is at least one spacer that tethers the liposomes in onelayer to the liposomes in an adjacent layer.

In one embodiment the method comprises measuring the refractive index ina volume enclosed by the bilayer structure.

In one embodiment the method comprises measuring the refractive index ina volume enclosed by liposomes tethered to the surface.

In one embodiment the method comprises measuring the refractive index ina volume enclosed by at least one liposome tethered to the surface.

In one embodiment the bilayer structure encloses a first volume of thedetection volume and wherein the volume not enclosed by the bilayerstructure but within the detection volume is a second volume and whereinthe ratio between the first and second volumes is optimised formeasurement of refractive index of the first volume. When it is desiredto measure the refractive index of the first volume the first volumeshould be as large as possible compared to the second volume. A largefirst volume will give higher sensitivity. Examples of suitable ratiosbetween the first volume and the second volume include but are notlimited to 0.001, 0.01, 0.1, 1, and 10. Also higher values of the ratioare encompassed by the present invention such as 20, 50, 100, 500, and1000.

In conclusion, the presently invented method to study cell-membranepermeation, advantageously admits direct measurements of the transferrate of both uncharged and charged solutes across biological membranes.The method is based on resolving the temporal change in refractive indexupon a permeation-dependent change in the solute concentration insideliposomes confined to the evanescent field associated with, for example,a surface plasmon resonance active sensor surface.

In a second aspect of the present invention there is provided a devicecomprising;

-   -   a) at least one surface,    -   b) at least one bilayer structure tethered to the surface, and    -   c) at least one sensor capable of detecting a change in        refractive index in a detection volume, wherein the bilayer        structure encloses a first volume of the detection volume and        wherein the volume not enclosed by the bilayer structure but        within the detection volume is a second volume and wherein the        ratio between the first volume and second volume is above about        0.001.

In alternative embodiments the ratio between the first volume and secondvolume is above about 0.001, preferably above about 0.01, morepreferably above about 0.1, even more preferably above about 1, and mostpreferably above about 10. A high ratio is desired because it leads to abetter sensitivity for the refractive index measurement in the firstvolume. One advantage of the present invention is that the sensitivityand accuracy can be increased by providing a high ratio.

In one embodiment the bilayer structure comprises at least one liposometethered to the surface. In yet another embodiment the bilayer structurecomprises a multitude of liposomes tethered to the surface.

In one embodiment the at least one liposome is tethered to the surfacewith at least one spacer. Examples of spacers include but are notlimited to a polymer, a nucleic acid, DNA, a His-tag, a biotinylatedlipid attached to avidin covalently bound to the surface, andhydrophobically modified dextran. Also any combination of the abovementioned spacers can be used.

In one embodiment the at least one spacer is a polymer. In anotherembodiment the at least one spacer is a His-tag. In a further embodimentthe there are both polymer spacers and His-tag spacers.

In one embodiment the distance between the bilayer structure and thesurface is less than about 250 nm.

In one embodiment the sensor which measures the refractive index in thedetection volume is selected from the group consisting of a surfaceplasmon resonance sensor, an ellipsometry sensor, and an opticalwaveguide laser spectroscopy sensor.

In one embodiment the sensor utilizes the phenomenon surface plasmonresonance.

DETAILED AND EXEMPLIFYING DESCRIPTION BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the successive addition of Neutravidin/Biotin DNA (N/B),0.1 M sucrose (S1), 2 mg/ml cholesterol DNA tagged POPC-liposomes(POPC), 0.1 M sucrose (S2), 40 μM melittin, 0.1 M sucrose (S3), 0.1 Msucrose (S4), 0.1 M sucrose (S5) to a PEG/PEG-biotin functionalizedBiacore® sensor.

FIG. 2 shows the response (RU) from the five sucrose additions (S1-S5).The insert shows a partial enlargement of FIG. 2.

FIG. 3 shows the uptake of sucrose into the liposomes as a function oftime, which is obtained by subtracting the change in RU upon addition ofsucrose to closed liposomes (S2) from the change in RU upon addition ofS3-S5.

FIGS. 4 a and 4 b show liposome permeability for glycerol, urea andhydroxyurea.

FIGS. 5 a and 5 b show the release and transport rate for glycerol overdifferent temperatures.

FIGS. 6 a to 6 c show the behaviour of cholesterol comprising liposomeswhen subjected to cyclodexterin.

FIG. 7 illustrates membrane transport of iodide and chloride ions.

EXAMPLE 1 Study of Melittin and its Effect on Membrane TransportMaterial and Methods

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phophocholine (POPC) was purchasedfrom Avanti Polar Lipids®. PEG-polymers (3 kDa), HS-PEG-NHCO-CH₂CH₂-OH(HS-PEG) HS-PEG-NHCO-CH₂CH₂-Biotin (HS-PEG-Biotin) were purchased fromRapp Polymere® GmbH. The Sensor chips were purchased from GEHealthcare®. The membrane protein analysis kit came from LayerLab®.HEPES-2 is 10 mM HEPES, 150 mM NaCl, pH 7.4.

Preparation of Liposomes

Liposomes were made by first evaporating the solvent, chloroform, fromthe POPC lipids using a flow of nitrogen gas. The dried lipids were thenkept under the flow of nitrogen gas for at least one hour after whichHEPES-2 was added to yield a 4 mg/ml concentration of POPC. Thedissolved POPC lipids were vortexed for at least 30 min to yieldmulti-laminar liposomes. Uni-laminar liposomes were produced using amini extruder and polycarbonate membranes with pore size of 50 nm fromAvanti Polar Lipids® with a 50 nm pore size. The radiuses of theliposomes had a Gaussian size distribution with a mean radius of 36 nm.The liposomes were stored under nitrogen gas at 4° C. until use (within2 days).

Functionalization of the Biosensor Surface

A mixture (300 μl) of HS-PEG (200 μg/ml) and HS-PEG-Biotin (20 μg/ml),dissolved in HEPES-2, were injected over the Biacore® Au sensor chip ata flow rate of 5 μl/min. Then a mixture (150 μl) of NeutrAvidin (40μg/ml) and Biotin-DNA (0.7 μM) was injected and bound to HS-PEG-Biotinat the surface.

Tethering of Liposomes

POPC liposomes (2 mg/ml, in HEPES-2) containing cholesterol-DNA tags (3DNA tags per liposome) were injected at a flow rate of 5 μl/min. The endof the DNA-part of the cholesterol-DNA tag is single-stranded andcomplementary in sequence to the Biotin-DNA at the surface enablingtethering of the liposomes to the sensor surface.

Melittin Binding to the Liposomes

The reversible binding of melittin was measured in the Biacore®instrument directly after the tethering of POPC liposomes was finished.Melittin solutions at a concentration of 0.5-40 μM in HEPES-2 wereinjected in sequence. During injection the melittin molecules bind tothe liposomes and after the injection is terminated, running buffer(HEPES-2) flows over the surface and melittin dissociates. After thedissociation was complete a new melittin solution of differentconcentration was injected.

Rupture of Liposomes by Melittin

Rupture of liposomes by melittin was measured in the Biacore® instrumentdirectly after the tethering of POPC liposomes. Melittin dissolved inHEPES-2 at a concentration of 360 μM (1 mg/ml) was injected and thedecrease in response (RU) as the liposomes rupture was monitored.

Melittin Mediated Uptake of Sucrose

After tethering of NeutrAvidin/Biotin-DNA the following injections weremade in sequential order; 0.1 M sucrose, 2 mg/ml cholesterol-DNA taggedPOPC-liposomes, 0.1 M sucrose, 40 μM melittin, 0.1 M sucrose, 0.1 Msucrose, 0.1 M sucrose. All samples were dissolved in HEPES-2.

RESULTS

Tethering of liposomes

There was binding of liposomes to the biosensor surface. The Carboxylicgroups at the surface were activated for amide binding using EDC/NHS.Thereafter PLL-PEG/PLL-PEG-Biotin (5:1), NeutrAvidin/Biotin-DNA (1:1)and cholesterol DNA tagged POPC-liposomes were added.

The bilayer structure enclosed a first volume of the detection volumeand the volume not enclosed by the bilayer structure but within thedetection volume is a second volume. The ratio between the first andsecond volumes was in this particular case about 0.1.

Melittin Binding and Pore Formation

Melittin solutions of various concentrations (0.5-40 μM) were injectedover the bound liposomes in the Biacore® instrument at 5 μl/min. Thechange in response (RU) as a function of time (t) was fitted toRU=RU₀+RU₂*(1−exp (−t/τ₁))+RU₂* (1−exp(−t/τ₂)). However, the change inRU during the injection of 0.5 μM melittin could be described using onlyone exponential (i.e. RU₂=0). It is known that high concentrations ofmelittin can rupture cells and liposomes. Unspecific binding (i.e.binding to the biosensor surface without liposomes) of melittin wassmall, less than 5% of the binding of melittin to the liposome membranesurface (data not shown). The observed rate constant, k₁, (k₁=1/τ₁) andthe response (RU₁), were studied as a function of the concentration ofmelittin in the bulk solution [Mel]_(B). The microscopic on-rate (k₊₁)and off-rate (k⁻¹) of melittin with the liposome membrane surface wereobtained from a fit of the data to k₁=[Mel]_(B)*k₊₁+k⁻¹, which gavek⁻¹=0.032 s⁻¹ and k₊₁=3684 M⁻¹s⁻¹. The amplitude, RU₁, was alsodetermined by the microscopic rate constants, RU₁=RU_(max)*(k₊₁/(k₊₁+k⁻¹). There was a good fit of RU₁ as a function of [Mel]_(B)when using k⁻¹=0.032 s⁻¹ and k₊₁=3684 M⁻¹s⁻¹ (which are obtained fromthe fit of k₁ as a function of [Mel]_(B)).

A comparison of the response (RU) from binding of liposomes to that frombinding of melittin, gives the number of melittin molecules that bindsto each liposome. It is known that the α-helical melittin molecule bindsto the membrane in a parallel orientation and the area of the liposomemembrane that a melittin molecule occupies is approximately 210 Å². Thesurface area of the liposome outer membrane is approximately 1.63*10̂6 Å²(liposome radius≈360 Å). Hence the concentration of melittin at theliposome outer membrane ([Mel]_(O.M.)) and the fraction (F_(O.M.)) ofthe liposome surface that the melittin molecules cover can be calculatedfor each [Mel]_(B).

At concentrations of 2, 4, 8 and 40 μM of melittin in the bulk solution,association of melittin with the liposome were biphasic. It is knownthat pore formation is induced at a threshold concentration of melittinat the membrane surface. If pores are formed, melittin molecules in thebulk solution outside the liposomes should be able to cross the liposomemembrane and bind to the inner membrane surface of the liposomes. It isthus assumed that the slower kinetic phase represent melittin binding tothe inner membrane. The observed rate constant, k₂, was found to belinear dependent on [Mel]_(B) which shows that transfer a melittinthrough the pore is not the rate limiting step (not dependent on[Mel]_(B)). A linear fit gave that k₊₂=56 M⁻¹s⁻¹ and that k⁻²=0.0046s⁻¹. The lower value of the apparent diffusion coefficient k₊₂ (56M⁻¹s⁻¹) compared to k₊₁ (3684 M⁻¹s⁻¹) can be explained by the lowerprobability of a melittin molecule finding the pores of the liposomethan the liposome surface as a whole. The number of pores/liposome isGaussian distributed and at low [Mel]_(O.M.) an increase in [Mel]_(B)increases the number of liposomes having one pore. Hence, at low[Mel]_(B) the response (RU₂) will not only depend on [Mel]_(B) but alsoon the fraction of liposomes that do not have any pores. At high[Mel]_(B), where all liposomes have at least one pore, the surfacecoverage of melittin at the inner membrane should equal that at theouter membrane, and RU₂ should approach RU_(2max)=RU₁*(Area_(In)/Area_(Out)). Hence the above results show that the slowerkinetic phase describes binding of melittin to the inner membrane.Further support comes from an observation that RU, after the injectionof 4, 8 and 40 μM is stopped, does not return to the initial value(pre-injection), which is explained by the melittin molecules beingtrapped inside the liposomes as the pores disappear due to melittindissociation from the membrane surface.

Melittin Pore Mediated Sucrose Uptake

A simple method for real-time and label-free uptake measurements ofanalytes across membranes at a biosensor surface would be of great valuewhen investigating for example membrane transporters. Here we wanted totest if it was possible to measure the uptake of an analyte, sucrose,through the melittin pore by simply detecting the change in refractiveindex of the solution inside the liposome caused by the analyte as it istaken up.

FIG. 1 shows the successive addition of Neutravidin/Biotin DNA (N/B),0.1 M sucrose (S1), 2 mg/ml cholesterol DNA tagged POPC-liposomes(POPC), 0.1 M sucrose (S2), 40 μM melittin, 0.1 M sucrose (S3), 0.1 Msucrose (S4), 0.1 M sucrose (S5) to a PEG/PEG-biotin functionalizedsurface.

FIG. 2 shows the response (RU) from the five sucrose additions (S1-S5).Adding sucrose changes the refractive index of the solution in thedetection volume, i.e. the volume corresponding to the evanescent fieldabove the sensor surface, which is manifested as a shift in RU. Thesmaller increase in RU upon adding S2 compared to adding S1 correspondsto the reduced accessible volume of the evanescent field caused by thesurface-bound liposomes. Addition of melittin produces pores in theliposomes membrane and hence makes the interior volume of the liposomesaccessible to the added sucrose molecules. As the melittin addition isstopped, melittin dissociates from the membrane surface and the numberof liposomes having melittin-pores decreases with time. FIG. 2 shows thechange in RU upon addition of sucrose as the number of pores/liposome isreduced (S3-S5).

FIG. 3 shows the uptake of sucrose into the liposomes as a function oftime, which is obtained by subtracting the change in RU upon addition ofsucrose to closed liposomes (S2) from the change in RU upon addition ofS3-S5. The amplitude of the uptake decrease from S3 to S5, which isexplained by a decreasing number of liposomes having pores. The ratealso decreases from S3 to S5, which is explained by decreasing number ofpores per liposome. From the graph in FIG. 3 the decay time for uptakeof sucrose can be estimated and τ₃≈20 sec. Given that the concentrationof sucrose inside the liposome after the uptake is equal to[sucrose]_(B)=0.1 M, then ˜7400 sucrose molecules cross the membrane in˜20 sec. Table 2 show that RU₂/RU_(2max) at [Mel]B=40 μM is 0.85 whichcorrespond to the number of liposomes having more than onepore/liposome. The amplitude of the response is in good agreement withthe value that can be expected from filling the liposomes with 0.1 M ofsucrose, which have a molecular weight (Mw) of 0.34 kDa. The expected RUof the 7400 sucrose molecules (0.1 M) inside the liposome can becalculated from the obtained response of immobilizing the liposomeitself (RU=6790). The Mw of the liposome is 35800 kDa, which gives 0.19RU/kDa. The Mw of 7400 sucrose molecules is 7400*0.342 kDa=2531 kDa,which gives that RU=2531*0.19=480. Since only ˜85% of the liposomes havepores the expected value is 480/0.85≈560 RU, which is in good agreementwith the measured response of 530 RU from the S3 injection.

EXAMPLE 2

SPR-Based Methods for Screening of Multiple Permeation Events byInvestigating the Permeability of the Non-Electrolytes Glycerol, Ureaand Hydroxyurea

The efficiency of the SPR-based method and its compatibility withscreening of multiple permeation events was evaluated by investigatingthe permeability of the non-electrolytes glycerol, urea and hydroxyurea,which are all biologically relevant molecules. For example, theefficiency by which glycerol is transported across the membranes of theadipocytes, where fat molecules are stored, has been suggested to be animportant factor in the development of obesity and type II diabetes[17]. Urea, on the other hand, is a waste product in the metabolicprocess and is transported out from liver cells and released from thebody via urine, while hydroxyurea is a drug that has been widely used incancer chemotherapy [18] as well as in treatment of HIV-virus infections[19]. The applicability of the method is demonstrated for in situalteration and simultaneous monitoring of liposome permeability bymonitoring the increase in glycerol permeation upon a gradualcyclodextrin-induced reduction in the liposomal cholesterol content.Also shown is the compatibility of the method to probe transport ofions.

At first 70 nm liposomes were provided in accordance with Example 1.FIG. 4 a shows binding of 70 nm liposomes followed by repeated injectionpulses of glycerol, urea, hydroxyurea at different concentrations (seeinset). FIG. 4 b shows a magnification of one of the rinsing steps, uponwhich the solute (glycerol) is released from the liposome interior. Alsoshown in FIG. 4 b (dashed curve) is an identical rinsing step withoutimmobilized liposomes, illustrating that the time constant of thefluidic exchange is faster than 100 ms, which is sufficient totemporally resolve these transport measurements. The rate of glycerolrelease was examined in a temperature interval from 8 to 22° C. Theresults are demonstrated in FIG. 5 a. In agreement with expectations,the rate of transport across the lipid membrane increases withincreasing temperature, and Arrhenius plots display a linear dependencebetween ln(1/) and 1/T, yielding activation energies in excellentagreement with literature data for the three solutes (glycerol, urea andhydroxyurea) investigated, see FIG. 5 b.

The results shown in FIGS. 4 and 5 demonstrate that the method can beused to accurately determine permeation coefficients of lipid membranesby following the release of solutes, rather than the uptake. Note thatall these data were obtained from a single experiment using the same setof immobilized liposomes. The reproducibility between differentexperiments was better than 2%.

70 nm liposomes containing 40% cholesterol were provided in accordancewith Example 1. Binding of the liposomes followed by repeated injectioncyclodextrin, which removes cholesterol from the lipid bilyaer of theliposomes is shown in FIG. 6 a. The SPR response is proportional to theinterfacial refractive index, which makes it possible to quantify theremoval of cholesterol. After seven injections of cyclodextrin, thecholesterol content was reduced to 14%.

Each injection of cyclodextrin (FIG. 6 a) was followed by an injectionpulse of glycerol, which enabled the time constant of glycerol transportversus cholesterol content to be quantified, ranging from around 7 s at40% cholesterol to 1 s at 14% cholesterol, see FIG. 6 b. Note that adifference in transport rate was detectable for as little as 1% changein cholesterol content.

FIG. 6 c shows the variations in permeability (estimated from the rateof solute transfer and the vesicle area) and the amplitude of theresponse (which is proportional to the internal volume). In agreementwith expectations, the permeability decreases linearly versuscholesterol content, while the internalized volume increasesnon-linearly. The latter observation reflects a structural change of themembrane at a cholesterol content of around 25%.

With the measurements shown in FIGS. 3 a to c, it is demonstrated thatthe method enables solute transfer to be quantified in terms ofpermeability, which can simultaneously be correlated with structuralchanges of the membrane properties. Information of this type is of highrelevance in studies on how, for example, drugs influence thepermeability of cell-membranes. Both passive and membrane-proteinmediated transfer of uncharged solutes is of high biological andpharmaceutical relevance. However, for a method like this to begenerically applicable for any type of membrane transport reaction, alsoion translocation should be possible to measure, which is todaytypically measured using patch-clamp based assays. FIG. 7 illustratesmembrane transport of iodide (˜10 s) and chloride (˜200 s) ions, inexcellent agreement with literature data. Note that gramicidin wasincorporated into the liposomes in order to ensure co-transport ofcounter ions (K+), thus prohibiting the establishment of anelectrostatic transmembrane potential that would otherwise restrict theunhindered transport of anions.

The results shown in FIG. 7 demonstrate the applicability of the methodto time resolve membrane translocation reactions also of chargedsolutes. The natural extension of this work is studies of molecule andion-channel controlled transport, of high relevance in thedrug-screening process.

As illustrated for glycerol, urea and hydroxyurea, the method accordingto the invention enables screening of multiple permeation events in asingle measurement, and allows for in situ alterations in permeabilityto be quantified, which is exemplified by successive removal ofcholesterol from the lipid bilayer. In comparison with alternativemethods to probe non-electrolyte transfer, which rely on indirectmeasurements osmotically-induced size changes of suspended liposomes,the method improves the sensitivity and reduces the required amount ofsample by orders of magnitude.

In the set of results shown in FIGS. 4 to 7, it is demonstrated thatsolute transport across the lipid bilayer membrane of liposomesimmobilized in an electromagnetic evanescent field can be determined byfollowing the changes in refractive index of the liposome-internalizedvolume upon solute release, rather than uptake (FIG. 4). It is alsodemonstrated that the membrane properties can be varied in situ, and becorrelated to changes in permeability (FIG. 5). In FIG. 6, wedemonstrate that not only uncharged solutes, but also the transport ofions can be measured by following changes in the refractive index of theliposome-internalized volume upon solute release (or uptake).

In summary, the present invention admits the possibility of using thissensing principle for direct measurements of molecular transport ofnon-electrolyte solutes across membranes with a time resolution of 100ms. The method is unique in the sense that it enables direct, ratherthan indirect (c.f. the DLS and fluorescence quenching methods),measurements of solute transfer across lipid bilayers, which was so farpossible only for charged molecules using patch-clamp [14] or chip-basedalternatives [15]. In comparison with the indirect methods, it alsooffers all other advantages of surface-based methods, such as rapidsequential (or parallel) screening of multiple solutes as well as insitu perturbation of liposome permeability by addition of effectormolecules. There is also no limitation with respect to the size of theanalyzed liposomes, while the DLS-based method is applicable onrelatively large liposomes only, since liposomes with a diameter smallerthan around 80 nm are essentially non-compressible (<1%) [16].

Although the invention has been described with regard to its preferredembodiments which comprise the best mode presently known to theinventors it should be understood that various changes and modificationsas would be obvious to one having the ordinary skill in this art may bemade without departing from the scope of the invention as set forth inthe claims appended hereto.

REFERENCES

-   1. E. Vierteljahrsschrift der Naturforschenden Gesellschaft in    Zürich.:. Overton, Vierteljahrsschrift der Naturforschenden    Gesellschaft in Zürich 40, 159 (1895).-   2. H. Danielli Davson, J. F., The Permeability of Natural Membranes.    (Cambridge University Press, Cambridge, England, 1943).-   3. P. Mueller, D. O. Rudin, H. T. Tien et al., Nature 194, 979    (1962).-   4. A. D. Bangham, Prog Biophys Mol Biol 18, 29 (1968).-   5. B. E. Cohen and A. D. Bangham, Nature 236 (5343), 173 (1972).-   6. A. S. Verkman, J Membr Biol 148 (2), 99 (1995).-   7. D. G. Levitt and H. J. Mlekoday, J Gen Physiol 81 (2), 239    (1983).-   8. P. Y. Chen and A. S. Verkman, Pflugers Arch 408 (5), 491 (1987).-   9. P. Y. Chen, D. Pearce, and A. S. Verkman, Biochemistry 27 (15),    5713 (1988).-   10. U. Jonsson, L. Fagerstam, B. Ivarsson et al., Biotechniques 11    (5), 620 (1991).-   11. L. S. Jung, C. T. Campbell, T. M. Chinowsky et al., Langmuir 14    (19), 5636 (1998).-   12. B. Liedberg, I. Lundstrom, and E. Stenberg, Sensors and    Actuators B-Chemical 11 (1-3), 63 (1993).-   13. M. Branden, S. Dahlin, and F. Hook, Chemphyschem 9 (17), 2480    (2008).-   14. E. Neher and B. Sakmann, Nature 260 (5554), 799 (1976).-   15. A. Janshoff and C. Steinem, Analytical and Bioanalytical    Chemistry 385 (3), 433 (2006).-   16. S. T. Sun, A. Milon, T. Tanaka et al., Biochimica Et Biophysica    Acta 860 (3), 525 (1986).-   17. E. M. Wintour and B. A. Henry, Trends Endocrinol Metab 17 (3),    77 (2006).-   18. C. Fausel, Am J Health Syst Pharm 64 (24 Suppl 15), S9 (2007).-   19. F. Lori, A. Foli, L. M. Kelly et al., Curr Med Chem 14 (2), 233    (2007).

1. A method for studying transport of an agent across a membranecomprising the steps of: a. providing at least one surface with abilayer structure tethered to the surface, said bilayer structurecomprising a detection volume, b. contacting the bilayer with at leastone agent to be analysed, and c. detecting a change in refractive indexin the detection volume resulting from transportation of the agentacross the membrane.
 2. The method according to claim 1, comprisingdetecting a change in refractive index with at least one sensor selectedfrom the group consisting of a surface plasmon resonance sensor, anellipsometry sensor, and an optical waveguide laser spectroscopy sensor.3. The method according to claim 1 wherein at least one entity selectedfrom the group consisting of a membrane protein, and an ionophore isassociated with the bilayer structure.
 4. The method according to claim1, wherein the bilayer structure comprises at least one liposome.
 5. Themethod according to claim 4, wherein at least one liposome is tetheredto the surface with a spacer molecule.
 6. The method according to claim1, wherein the bilayer structure comprises at least two layers ofliposomes.
 7. The method according to claim 1, comprising measuring therefractive index in a volume enclosed by the bilayer structure.
 8. Themethod according to claim 1, wherein the bilayer structure encloses afirst volume of the detection volume and wherein the volume not enclosedby the bilayer structure but within the detection volume is a secondvolume and wherein the ratio between the first and second volumes isoptimised for measurement of refractive index of the first volume.
 9. Adevice comprising a. at least one surface, b. at least one bilayerstructure tethered to the surface, and c. at least one sensor capable ofdetecting a change in refractive index in a detection volume, whereinthe bilayer structure encloses a first volume of the detection volumeand wherein the volume not enclosed by the bilayer structure but withinthe detection volume is a second volume and wherein the ratio betweenthe first volume and second volume is above about 0.001.
 10. The deviceaccording to claim 9, wherein at least one entity selected from thegroup consisting of a membrane protein, and an ionophore is associatedwith the bilayer structure.
 11. The device according to claim 9, whereinthe bilayer structure comprises at least one liposome tethered to thesurface.
 12. The device according to claim 11, wherein at least oneliposome is tethered to the surface with at least one spacer.
 13. Thedevice according to claim 12, wherein the at least one spacer is atleast one selected from the group consisting of a polymer, a nucleicacid, DNA, a His-tag, a biotinylated lipid attached to avidin covalentlybound to the surface, and hydrophobically modified dextran.
 14. Thedevice according to claim 9, wherein the sensor has the capability tomeasure changes in refractive index in the interval 0-250 nm from thesurface.
 15. The device according to claim 9, wherein the sensor isselected from the group consisting of a surface plasmon resonancesensor, an ellipsometry sensor, and an optical waveguide laserspectroscopy sensor.
 16. The device according to claim 9, wherein thesensor utilizes the phenomenon surface plasmon resonance.